Transgenic expression of FoxM1 promotes endothelial repair following lung injury induced by polymicrobial sepsis in mice

Abstract

Enhancing endothelial barrier integrity for the treatment of acute lung injury (ALI) is an emerging novel therapeutic strategy. Our previous studies have demonstrated the essential role of FoxM1 in mediating endothelial regeneration and barrier repair following lipopolysaccharide-induced lung injury. However, it remains unclear whether FoxM1 expression is sufficient to promote endothelial repair in experimental models of sepsis. Here, employing the FoxM1 transgenic (FoxM1 Tg) mice, we showed that transgenic expression of FoxM1 promoted rapid recovery of endothelial barrier function and survival in a clinically relevant model of sepsis induced by cecal ligation and puncture (CLP). We observed lung vascular permeability was rapidly recovered and returned to levels similar to baseline at 48 h post-CLP challenge in FoxM1 Tg mice whereas it remained markedly elevated in WT mice. Lung edema and inflammation were resolved only in FoxM1 Tg mice at 24 h post-CLP. 5-bromo-2-deoxyuridine incorporation assay revealed a drastic induction of endothelial proliferation in FoxM1 Tg lungs at 24h post-CLP, correlating with early induction of expression of FoxM1 target genes essential for cell cycle progression. Additionally, deletion of FoxM1 in endothelial cells, employing the mouse model with endothelial cell-restricted disruption of FoxM1 (FoxM1 CKO) resulted in impaired endothelial repair following CLP challenge. Together, these data suggest FoxM1 expression in endothelial cells is necessary and sufficient to mediate endothelial repair and thereby promote survival following sepsis challenge.

Figure 1. Rapid recovery of vascular permeability in FoxM1 Tg lungs following CLP challenge.

(A) Representative Western blotting demonstrating increased expression of FoxM1 in FoxM1 Tg lungs. Thirty µg of lung lysates were loaded per lane. FoxM1 expression was detected with anti-FoxM1 antibody. The same membrane was immunoblotted with an anti-β-actin antibody for loading control. (B) Lung vascular permeability assessed by EBA extravasation assay. Various times following CLP challenge, mouse lung tissues were collected for EBA assay. Lung tissues from sham-operated mice at 24 h post-surgery were collected as controls. Data are expressed as mean ± SD (n = 3–5 per group). *, P<0.01 versus FoxM1 Tg. (C) Lung wet/dry weight ratio. At 24 h post-surgery, lung tissues were collected and dried at 60°C for 3 days. Data are expressed as mean ± SD (n = 4). *, P<0.05 versus WT.

Figure 2. Accelerated resolution of lung inflammation in FoxM1 Tg mice.

(A) MPO activities in lung tissues. Lung tissues at indicated times post-CLP challenge were collected for MPO activity determination. Lung tissues from sham-operated mice at 24 h post-surgery were collected as controls. Data are expressed as mean ± SD (n = 3–5). *, P<0.001 versus WT; **, P<0.05 versus WT. (B) Representative micrographs of H & E staining of lung sections. At 24 h post-surgery, lungs were fixed for sectioning and H & E staining. Arrows indicate perivascular leukocyte infiltration. Scale bar, 50 µm.

Figure 3. Normalized expression of proinflammatory cytokines and adhesion molecule in FoxM1 Tg lungs at 24 h post-CLP.

RNA were isolated from lungs collected at 24 h post-surgery and QRT-PCR analysis were employed to assess the expression levels. Data are expressed as mean ± SD (n = 3–4). *, P<0.05 versus WT-sham.

Figure 4. Increased survival of FoxM1 Tg mice following CLP challenge.

3 month old mice were monitored for 7 days to determine the survival rate following CLP challenge (n = 13 WT and 14 FoxM1 Tg). Sham-operated mice (n = 5 WT or FoxM1 Tg) were also monitored for survival. *, P<0.001 versus CLP-WT. Tg, FoxM1 Tg.

Figure 5. FoxM1-induced endothelial cell proliferation in FoxM1 Tg lungs following CLP challenge.

(A) Representative micrographs of immunofluorescent staining. Lung tissues were collected at 24 h post-CLP challenge, sectioned and immunostained with anti-BrdU (green) and anti-vWF and CD31 (red) antibodies. Nuclei were counterstained with DAPI (blue). Arrows indicate proliferating EC. Scale bar, 50 µm. (B) Quantification of BrdU-positive nuclei. Data are expressed as mean ± SD (n = 4 per group). *, P<0.001 versus WT. (C) Quantification of BrdU-positive EC (vWF⁺ or CD31⁺) and non-EC (vWF^- or CD31⁻). BrdU-positive EC were quantified in small vessels (diameter ≤ 100 µm) and capillaries. Data are expressed as mean ± SD (n = 4). P<0.001 versus WT.

Figure 6. Early induction of expression of FoxM1 target genes essential for cell cycle progression in FoxM1 Tg lungs.

(A–D) QRT-PCR analysis of expression of FoxM1 target genes. Lung tissues were collected at indicated times post-CLP challenge or 24 h post-sham operation for RNA isolation and QRT-PCR analysis. Data are expressed as mean ± SD (n = 3–5 per group). *, P<0.001 versus WT; **, P<0.05 versus WT. (E) Representative Western blotting demonstrating FoxM1-mediated induction of Cdc25C protein expression. Lung tissues were collected at various times post surgery and lysed for examination of Cdc25C protein levels by Western blotting. The same membrane was blotted with anti-β-actin as a loading control. The experiment was repeated three times with similar data.

Figure 7. Time course of FoxM1 expression in lungs following CLP challenge.

Lung tissues were collected at indicated times post-CLP challenge or 24 h post-sham surgery for RNA isolation and QRT-PCR analysis. Expression of endogenous mouse FoxM1 was assessed with the primers specific with mouse FoxM1 (A) whereas expression of the transgene was analyzed with the primers specific with human FoxM1 (B). Data are expressed as mean ± SD (n = 3–5 per times). *, P<0.01 versus Sham; **, P<0.001. Mouse FoxM1 was similarly induced in WT or FoxM1 Tg lungs following CLP challenge but not in FoxM1 CKO lungs. Human FoxM1 was constitutively expressed in FoxM1 Tg lungs at various times post-CLP.

Figure 8. Impaired endothelial repair in FoxM1 CKO lungs following CLP challenge.

(A) EBA extravasation assay demonstrating sustained vascular leakiness in FoxM1 CKO lungs. Data are expressed as mean ± SD (n = 4–5). *, P<0.01 versus WT-18 h; **, P<0.005 versus WT-18 h; #, P>0.5 versus FoxM1 CKO-18 h. (B) Persistent increase of MPO activity in FoxM1 CKO lungs following CLP challenge. Data are expressed as mean ± SD (n = 4–5). *, P<0.01 versus WT-18 h; **, P<0.005 versus WT-18 h; #, P>0.5 versus FoxM1 CKO-18 h.